Understanding living systems requires to be able to observe the dynamics of cellular proteins in real-time with a very high resolution both in space and time. In this context, fluorescence has become the read-out of choice for observing or detecting proteins in living cells or living organisms.
The selective detection of a protein of interest by fluorescence microscopy requires the presence of a fluorescent probe that acts as a contrast agent. This probe can be intrinsic, i.e. be part of the native protein, or extrinsic, i.e. specifically attached to the protein. Most of the time, a protein of interest does not possess a specific intrinsic probe that can give a unique optical signature for fluorescence imaging. Therefore, functionalization of the protein with a fluorescent probe displaying photophysical properties not present in the biological system under study ensures a specific optical signature. The method of choice for obtaining a specific labeling in living cells is to fuse the protein of interest to an additional polypeptide, a so-called tag, which can become fluorescent by an autocatalytic process generating a fluorophore, as in the green fluorescent protein (GFP), or act as an anchor for the specific and tight (covalent or non-covalent) binding of a fluorophore.
In this context, an ideal fluorescent protein-based probe must (i) not perturb the function of the protein it is attached to, which includes not interfering with the overall folding of the native protein, not interfering with the localization of the protein, not changing its interaction with partners, (ii) be functional in the cellular compartment where the protein acts no matter what the pH or redox conditions are, and in the conditions of living of the cells or organisms under study, (iii) enable the imaging of the protein with time resolution compatible with the time scales found among biological processes. This latter point means that ideally a protein-based fluorescent probe must (i) be fluorescent as soon as the fusion protein is folded to report on early stage of the protein life, (ii) be photostable on long-term to allow for quantitative observation of proteins involved in slow dynamic cellular processes. This latter point is particularly important as quantitative observation of fluorescently labeled proteins by fluorescence microscopy is often limited by the photobleaching of the fluorophore. Indeed, as organic fluorophores can only experience a limited number of excitation-emission photocycles resulting in the production of only a limited number of photons before being photochemically destroyed, continuous observation longer than few seconds or few minutes is in general impossible in fluorescence microscopy, which is particularly problematic for long-term tracking or single-molecule imaging.
Specific fluorescent labelling of a protein of interest in living cells is generally achieved through the use of an additional polypeptide, a so-called tag, which is fused to the protein of interest. This is easily done with the currently available DNA recombinant technology: the DNA sequence encoding the additional polypeptide is cloned in frame with the DNA sequence encoding the protein of interest. The resulting DNA sequence encodes a chimeric protein resulting from the fusion of the two polypeptides.
Among the known technologies, the additional peptidic tag can become fluorescent by itself through an autocatalytic process. This is the case for the green fluorescent protein (GFP) identified in the jellyfish Aequorea Victoria. The covalent chromophore of GFP, the parahydroxybenzylidene-5-imidazolinone (p-HBI) results from the cyclization/dehydration/oxidation of a triad Gly/Tyr/Ser in the protein backbone. This approach benefits from the fully genetically encoding of the fluorophore as the chromophore forms from the peptidic sequence of GFP. There is therefore an absolute specificity of the fluorescent labeling when using GFP as a fluorescent probe. Several GFP-like proteins have been discovered or engineered to obtain a collection of GFP-like proteins with photophysical properties in the whole visible spectrum. However, this approach suffers from several limitations. First, the autocatalytic maturation of the fluorophore within the protein beta-barrel is a slow multi-step process with a half-time between 40 minutes and 2 hours for most fluorescent proteins (Chudakov et al., 2010): there is thus a lag time between the end of the protein folding and the appearance of the fluorescence, which prevent the study of early stage of the protein life. The second limitation is that molecular O2 is necessary during the oxidation steps involved in the fluorophore formation, which limits the use of such GFP-like fluorescent proteins to environments with O2 (>3 μM) (Hansen et al., 2001), and prevents their use as reporters in anoxic or hypoxic biological systems. The third limitation is the photostability of the GFP-like fluorescent proteins. The photobleaching halftime (required to reduce the emission rate to 50% from an initial emission rate of 1,000 photons/s per fluorescent protein) for common GFP-like fluorescent proteins is typically between 5 and 200 s (Shaner et al., 2005). Finally, the size of the GFP-like fluorescent proteins, comprised between 25 and 30 kDa, has been shown in some cases to modify the function of the protein it is fused to.
Alternative fluorescent proteins relying on a small apo-protein that strongly binds covalently or non-covalently an endogenous fluorogenic cofactor are also used. The binding leads to an exaltation of the cofactor brightness. Fluorescence exaltation results from constraining the fluorogenic cofactor within a particular conformation, favoring radiative deexcitation. Relying on the binding of an endogenous cofactor enables to overcome the necessity for molecular oxygen allowing working under anoxic conditions. Moreover, most of the known alternative cofactor-based fluorescent proteins are much smaller than GFP, which is an advantage to minimize the risk of perturbing the function of the studied protein.
Among these alternative fluorescent proteins, bacteriophytochromes binding covalently biliverdin were engineered into near-infrared fluorescent proteins such as IFP1.4 (Shu et al., 2009), iRFP (Filonov et al., 2011) and Wi-Phy (Auldridge et al., 2012). However, these proteins have a long maturation time (halftime 2 hours) because they require the covalent attachment of the biliverdin cofactor. Moreover, they are multimeric, which can modify the function and localization of the studied protein. Finally they show photostability half time between 50 and 450 s, which is of the same order of magnitude than the GFP-like fluorescent proteins.
Flavin mononucleotide (FMN)-binding green fluorescent proteins were engineered from Light-oxygen-voltage-sensing (LOV) domains (Chapman et al., 2008; Drepper et al., 2007; Shu et al., 2011). These proteins bind non-covalently FMN with subnanomolar affinity. They have been shown to be a good substitute of GFP for studies in anaerobic conditions.
Recently, a green fluorescent protein called UnaG was identified from unagi eel (Kumagai et al., 2013); its fluorescence results from the non-covalent tight binding of bilirubin with subnamolar affinity. The formation of the fluorescent protein is fast and does not require oxygen.
In parallel of these developments, a hybrid concept has been proposed based on engineered single-chain antibodies acting as fluorogen-activating proteins (FAP), which bind noncovalently well-known synthetic fluorogens (mostly molecular rotors and cyanines) (Ozhalici-Unal et al., 2008; Shank et al., 2009; Szent-Gyorgyi et al., 2008). Single-chain antibody (scFv) reporters generating fluorescence from otherwise weakly fluorescent thiazole orange and malachite green have been isolated by screening scFvs libraries by fluorescence activating cell sorting (FACS) (Szent-Gyorgyi et al., 2008). However ScFv-based FAP contains disulfide linkages, and are therefore adapted only for use in non-reducing environments, such as the cell surface and the secretory apparatus.
However, there is still a need in the art for a tunable protein-based reporting system for imaging fusion proteins comprising a protein of interest fused to a fluorescent tag in living cells and in multi-cellular organisms, wherein said reporting system implies a highly dynamic binding to a fluorogen, thereby allowing the fluorescence to be switched on and off rapidly by addition or withdrawing of the fluorogen, opening new opportunities for multiplexing imaging.